Cleavage experiments with deoxythymidine 3′,5′‐bis‐(p‐nitrophenyl phosphate) suggest that the homing endonuclease I‐PpoI follows the same mechanism of phosphodiester bond hydrolysis as the non‐specific Serratia nuclease1

Friedhoff, Peter; Franke, Ingo; Krause, Kurt L.; Pingoud, Alfred

doi:10.1016/s0014-5793(98)01660-3

Cited by 38 publications

(40 citation statements)

References 17 publications

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“…Using synthetic substrates [134], site-directed mutagenesis [166,167] and crystal structures [177,183], Friedho¡ et al [210] proposed a`general base model' for the reaction mechanism of Serratia nuclease (Fig. 9A).…”

Section: Water-assisted Metal Ion Catalysismentioning

confidence: 99%

“…It has been postulated that Asn119 is probably involved in the binding and correct positioning of the metal cofactor. Arg57 has been shown to be involved in the transition state stabilization [210]. Interestingly, homing endonuclease I-PpoI from Physarum polycephalum [210,211], colicin E9 endonuclease [212] and junction resolvase endonuclease (Endo VII) from phage T4 cells [213] shows an identical active site and has been postulated to have a similar mechanism.…”

Section: Water-assisted Metal Ion Catalysismentioning

confidence: 99%

“…Arg57 has been shown to be involved in the transition state stabilization [210]. Interestingly, homing endonuclease I-PpoI from Physarum polycephalum [210,211], colicin E9 endonuclease [212] and junction resolvase endonuclease (Endo VII) from phage T4 cells [213] shows an identical active site and has been postulated to have a similar mechanism. Anabaena nuclease follows a similar reaction mechanism, where His124 acts as the general base, Asn155 in binding and correct positioning of the cofactor, and Glu163 in protonation of the leaving group (Fig.…”

Section: Water-assisted Metal Ion Catalysismentioning

confidence: 99%

“…Although Lunin et al [178] initially proposed a general acid model for Serratia nuclease, subsequently based on the observation of Kolmes et al [134] that the E127A mutant, and not H89A, cleaves deoxythymidine 3P,5P-bis-(p-nitrophenyl) phosphate (as the protonation of the leaving group is not necessary), Shlyapnikov et al [179] proposed a consensus mechanism similar to the one proposed by Friedho¡ et al [210]. In the case of Serratia nuclease, the rate determining step for the hydrolysis of oligonucleotides is substrate binding and/or phosphodiester bond cleavage but not product dissociation [133].…”

Section: Water-assisted Metal Ion Catalysismentioning

confidence: 99%

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Sugar non-specific endonucleases

Rangarajan¹

2001

FEMS Microbiology Reviews

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Sugar non-specific endonucleases are multifunctional enzymes and are widespread in distribution. Apart from nutrition, they have also been implicated in cellular functions like replication, recombination and repair. Their ability to recognize different DNA structures has also been exploited for the determination of nucleic acid structure. Although more than 30 non-specific endonucleases have been isolated to date, very little information is available regarding their structure^function correlations except that of staphylococcal and Serratia nucleases. However, during the past few years, the primary structure, nature of the active site based on sequence homology, and the probable mechanism of action have been postulated for some of the enzymes. This review describes the purification, characteristics, biological role and applications of sugar non-specific endonucleases. ß

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Section: Water-assisted Metal Ion Catalysismentioning

confidence: 99%

Section: Water-assisted Metal Ion Catalysismentioning

confidence: 99%

Section: Water-assisted Metal Ion Catalysismentioning

confidence: 99%

Section: Water-assisted Metal Ion Catalysismentioning

confidence: 99%

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Sugar non-specific endonucleases

Rangarajan¹

2001

FEMS Microbiology Reviews

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“…The proposed catalytic mechanism for I-PpoI/Serratia endonucleases concerns the direct contact of the pro-S P -nonbridging oxygen and the 3'-bridging oxygen with hydrated magnesium ion [3]. Elemental replacement of any of these two oxygens by sulfur destroys the architecture of the active site, enabling the attack of a water molecule activated by the histidine residue located at the face opposite to the scissile P-O 3' bond ( Fig.…”

mentioning

confidence: 99%

Stereochemical support for Serratia endonuclease active-site geometry

Stec¹,

Koziołkiewicz²,

Taira³

2000

Pure and Applied Chemistry

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Earlier observation that stereoregular All-R p -PS-oligonucleotides in the presence of nonsequence-specific Serratia endonuclease undergo nucleolytic degradation, confronted with recently published results on the active-site architecture of this enzyme, strongly supports an involvement of 3′-bridging and pro-Sp-nonbridging oxygen atoms of scissile internucleotide bond in the interactions with hydrated magnesium ion anchored by Asn-119 residue of this endonuclease.Recently, the similarity between the active site of I-PpoI specific endonuclease and Serratia marcescens nonsequence-specific endonuclease was pointed out by two independent groups [1,2]. The different folding of these two proteins determines the specificity requirement, but both proteins contain conserved essential asparagine and histidine residues at their active sites and exhibit a similar one metalion co-ordination pattern. Therefore, all main active-site components required for the cleavage of the internucleotide phosphorus-oxygen bond are in the similar arrangement.The general-base histidine 98 (I-PpoI) or 89 (Serratia) residue activates a water molecule for its nucleophilic attack at phosphorus and this occurs opposite from the site of the leaving 3'-oxygen. The ligand for the protein is comprised of the magnesium ion surrounded by three water molecules and Asn-119. Upon binding of the protein, the magnesium ion stabilizes the transition state by coordinating two oxygens of scissile phosphate, namely the 3'-oxygen of P-O-C 3' bond and the pro-Sp-nonbridging oxygen ( Fig. 1A; a) [3]. This magnesium ion does not coordinate a water molecule for nucleophilic attack at phosphorus opposite the leaving 3'-oxygen. Such architecture of the active site of the Serratia marcescens endonuclease comprising coordination of two oxygen atoms of the same scissile phosphate to the metal ion seems to be more plausible than the alternate mechanism for the mode of action of Serratia enzyme discussed by Miller et al. [4] and presented in Fig. 1B. The metal-bound water is deprotonated by a general base and attacks the phosphorus atom of the negatively charged phosphate, coordinated to the same metal ion through the 3'-bridging oxygen.Neither of two nonbridging oxygens of phosphate moiety contacts the metal ion. Such a model of active site was also preferred by Flick et al. [5], discussing the mode of action of homing endonuclease I-PpoI. Our choice of the mechanism as presented in Fig. 1A is based upon arguments concerning the stereoselectivity of Serratia enzyme towards stereoregular oligo(nucleoside phosphorothioate)s [6,7]. Since 1995, it has been known that Serratia marcescens endonuclease is stereoselective toward oligo(nucleoside phosphorothioate)s and cleaves only internucleotide phosphorothioates of R P configuration ( Fig. 1A; b) [6]. Although that cleavage required 10 3 higher concentration of enzyme than that effective for the cleavage of isosequental PO-oligonucleotide, a further increase in enzyme concentration does not cause the cleavage of any ...

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Effects of dimerization of Serratia marcescens endonuclease on water dynamics

et al. 2006

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The dynamics and structure of Serratia marcescens endonuclease and its neighboring solvent are investigated by molecular dynamics (MD). Comparisons are made with structural and biochemical experiments. The dimer form is physiologic and functions more processively than the monomer. We previously found a channel formed by connected clusters of waters from the active site to the dimer interface. Here, we show that dimerization clearly changes correlations in the water structure and dynamics in the active site not seen in the monomer. Our results indicate that water at the active sites of the dimer is less affected compared with bulk solvent than in the monomer where it has much slower characteristic relaxation times. Given that water is a required participant in the reaction, this gives a clear advantage to dimerization in the absence of an apparent ability to use both active sites simultaneously.

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Cleavage experiments with deoxythymidine 3′,5′‐bis‐(p‐nitrophenyl phosphate) suggest that the homing endonuclease I‐PpoI follows the same mechanism of phosphodiester bond hydrolysis as the non‐specific Serratia nuclease¹

Cited by 38 publications

References 17 publications

Sugar non-specific endonucleases

Sugar non-specific endonucleases

Stereochemical support for Serratia endonuclease active-site geometry

Effects of dimerization of Serratia marcescens endonuclease on water dynamics

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